Attenuated mutant newcastle disease virus strains for in ovo vaccination, method for preparing and their use

ABSTRACT

The present invention relates to new attenuated mutant New Castle&#39;s disease La SotaNewcastle disease virus strains. In particular, it relates to an attenuated mutant Newcastle&#39;s disease La SotaNewcastle disease virus strain suitable for in ovo vaccination of avian species comprising a mutation in the gene sequences encoding the HN and/or F glycoproteins of said virus resulting in an altered expression of said glycoproteins. Furthermore, the invention relates to a vaccine composition comprising said attenuated mutant Newcastle&#39;s disease La Sota virus strain, and to the use of said attenuated mutant Newcastle&#39;s disease La Sota virus strain for the preparation of a vaccine for in ovo vaccination of avian species against Newcastle&#39;s disease. Finally, the present invention also concerns a method for producing a vaccine for in ovo vaccination of avian species, said vaccine comprising attenuated mutant avian virus strains, which are selected using virus specific antibodies.

FIELD OF THE INVENTION

[0001] The present invention concerns new attenuated mutant Newcastledisease virus strains. The invention includes a more general method forselecting attenuated virus strains based on the use of virus specificantibodies, and particular antibodies specific for Newcastle disease LaSota virus. The invention also includes the use of said attenuatedmutant Newcastle disease virus La Sota strains in a vaccine for in ovovaccination of avian species, preferably chickens.

BACKGROUND

[0002] Antibodies are powerful tools for analyzing mutations in antigens(Pollock et al. 1987) and they have been successfully used for selectingantigenic variants, also known as escape mutants, of influenza virus(Gerhard and Webster 1978, Lubeck et al. 1980, Yewdel et al. 1986),rabies virus (Wiktor and Koprowski 1980) and measles virus (Birrer etal. 1981). NDV escape mutants were produced by Russel (1.983), Abenes etal. (1986), Meulemans et al. (1987) and Yussof et al. (1989). Meulemanset al. (1987) showed that NDV escape mutants may be more, or lesspathogenic than the parental virus strains (Meulemans et al. 1987).

[0003] The production of antibody resistant virus strains todeliberately attenuate the parental virus and make it suitable as in ovovaccine was never suggested. Benejean et al. (EP0583998) attenuated therabies virus by producing an escape mutant and used it as a vaccine.They never suggested or implied the use of this technique to produceescape mutants of avian viruses possibly useful for in ovo vaccination.

[0004] In ovo vaccination'technology using approved vaccine is a safe,efficacious, and convenient method for vaccination of poultry (Ricks etal. 1999, U.S. Pat. No. 6,032,612, A01K45/00C). In 1999, more than 80%of the U.S. broiler industry had converted to the in ovo vaccinationprocess to control Marek's disease (Ricks et al. 1999).

[0005] Studies within the last few years have shown that only few livevaccines that are routinely administered to hatched chickens may also beinjected into embryonated eggs during late stages of embryonationwithout a toxic effect. The turkey herpes virus (HVT, Sharma andBurmester, 1982), and infectious bursal disease virus (IBDV) strains oflow virulence (Sharma, 1985) can be used as embryo vaccines to induceactive protection against the homologous strains.

[0006] IBDV strains of moderate virulence such as 2512 (Sharma 1985),commercial infectious bronchitis virus (IBV) strains such asMassachusetts 41 (Wakenell and Sharma 1986) and Newcastle disease virusstrains such as the B1 (Ahmad and Sharma, 1992, 1993) and the La Sotastrain, cannot be employed for in ovo vaccination in their current formdue to embryonic toxicity. Attenuation of the virus strains currentlyused for post hatch vaccination is thus required to obtain strains withreduced pathogenicity to the avian embryo.

[0007] Wakenell and Sharma (1986) reduced the pathogenicity to theembryo of the Massachusetts 41 IBV strain using a tissue cultureattenuation system. At the 40^(th) passage in chicken kidney tissueculture, the virus became apathogenic for the embryos and embryonicvaccination induced IBV specific antibody production and protectionagainst virulent Massachusetts 41 IBV at 4 weeks of age.

[0008] Treatment of the B1 strain of NDV with the alkylating agentethylmethane sulfonate markedly reduced the virulence of this strain forthe 18-day chick embryo, and in ovo vaccination with this strainresulted in NDV specific antibody production and protection againstchallenge with the Texas GB strain (Ahmad and Sharma 1992).

[0009] Further, it was claimed (EP0848956 A1) that a vaccine preparationcontaining Newcastle disease viruses of the strain NDW was particularlysuited for in ovo application.

[0010] Finally, Mebatsion and Schrier (EP1074614A1) produced a NDV LaSota mutant, which is suited as vaccine candidate for in ovovaccination. The mutant expresses reduced levels of V protein and cansafely be administered to chicken embryos before hatch. Noantibody-based selection was used to obtain these strains.

[0011] The formation of a complex between a measured amount of antibodywith IBDV neutralizing activity and a specific amount of IBDVneutralized the pathogenicity of the IBDV and made it useful as in ovovaccine (U.S. Pat. No. 5,871,748, Whithfill et al. 1992, 1995, Haddad etal. 1997).

[0012] In order to reduce the economic losses due to Newcastle diseasein the commercial poultry industry, chickens currently have to bevaccinated against the Newcastle disease virus. It may be advantageousto use embryo vaccination for said purpose, in particular since in ovoinjection can be done using semiautomatic machines with multipleinjection heads allowing individual vaccination.

[0013] However, many vaccines used conventionally for post-hatchvaccination of birds cannot be used for in ovo vaccination. It istherefore an aim of the present invention to provide attenuatedNewcastle disease virus strains which can be effectively used as avaccine in avian species, administrable post hatch or in ovo. Anotheraim of the present invention is to provide a general method forselecting attenuated avian virus strains, and in particular attenuatedstrains of Newcastle disease virus.

DESCRIPTION

[0014] According to a first aspect, the present invention relates to anattenuated mutant Newcastle disease La Sota virus strain suitable for inovo vaccination of avian species comprising a mutation in the genesequences encoding the HN and/or F glycoproteins of said virus resultingin an altered expression of said glycoproteins. The term ‘attenuatedstrain’ relates to a strain which is less virulent than the parentalstrain.

[0015] La Sota is a lentogenic Newcastle disease virus strain. Severalpathotypes of Newcastle disease virus have been identified, i.e.velogenic, mesogenic and lentogenic. Although these terms result fromlaboratory tests carried out both in vivo and in vitro, the terms arenow generally used to describe viruses of high, moderate or lowvirulence for chickens. The neurotropic velogenic form of the disease iscaused by highly pathogenic strains of Newcastle disease virus and ischaracterised by a sudden onset of severe respiratory signs followed byneurological signs. In most cases the infected animals do not survive.Viscerotropic velogenic Newcastle disease virus strains are also highlypathogenic and cause high mortality and severe lesions in thegastrointestinal tract. Mesogenic strains of Newcastle disease virususually cause severe respiratory disease in fully susceptible birds, andin adult birds cause a marked drop in egg production. Lentogenic strainsof Newcastle disease virus cause generally a mild disease which ischaracterised by respiratory signs, especially in young fullysusceptible birds.

[0016] The attenuated mutant Newcastle disease La Sota virus strains ofthis invention are suitable for in ovo vaccination of any avian animal,whether domestic or wild, and particularly those which are commerciallyreared for meat or egg production. Without limitation thereto, exemplaryavian species include chickens, turkeys, pigeons, pheasants, and thelike. Birds, which are reared in high density brooder houses such asbroiler and layer chickens, are especially vulnerable to environmentalexposure to infectious agents and would largely benefit from pre hatchvaccination. Preferably, chickens, turkeys and pigeons are used.

[0017] As will be explained further below in this description andparticularly in example 1, HN and F antigenic variant virus strains wereobtained from the lentogenic La Sota Newcastle disease strain by aprocess called immunoselection, using the Mabs 8C11 (Le Long et al.1986) and 1C3 (Le Long et al. 1988) directed against the F and the HNglycoproteins of Newcastle disease, respectively. Four strains wereobtained in total. The F and HN strains were selected with monoclonalantibodies 1C3 and 8C11, respectively. In addition, two double mutantswere produced by immunoselection using F and HN specific monoclonalantibodies 1C3 and 8C11 starting from the HN and F mutant strains,respectively.

[0018] These four attenuated Newcastle disease La Sota virus strainswere further characterized in haemagglutination inhibition and ELISAtests and by sequence analysis of the genes coding for the F- and theHN-glycoproteins. These results are described in Table 3, 4 and 5 ofexample 2. Said characterization revealed that the different NDV-strainsselected with the monoclonal antibody 1C3 are characterized by asubstitution of amino acid 72 of the F gene. Indeed, the F-gene of the Fand F+HN mutant strains selected using the monoclonal antibody 1C3differ from the parental La Sota strain by a point mutation G-to-T (GATto TAT) leading to an Asp-72-Tyr substitution. Immunoselection of theHN-mutant, characterized by a Arg-101-Met substitution in the F gene,with 1C3 to obtain the HN+F mutant resulted in an additional Asp-Glusubstitution at this same amino acid 72. The Asp-72-Tyr substitution wasobserved earlier in a 1C3-resistant mutant of the Beaudette strain(Yusoff et al. 1989) while an Asp-72-Gly transition was observed in1C3-resistant mutants of the Italien NDV strain (Neyt et al. 1989), theBeaudette C strain (Yusoff et al., 1989) and in an antigenic variant ofthe Sato strain (Toyoda et al. 1988). Determination of thethree-dimensional structure of the fusion protein of NDV showed that theinvolved 1C3-epitope is surface exposed and situated at the loop segmentbetween strand 111a and the interchain disulfide (Chen et al. 2001) atantigenic sites A1, II and 1. Site A1 is as defined by Yusoff et al.(1989), 11 is as defined by Toyoda et al. (1988) and 1 is as defined byNeyt et al. (1989). Table 4 illustrates that the F−, F+HN− and HN+Fmutants did not only lose the 1C3-epitope, but also the 10F2-epitopesituated on the same loop (Chen et al. 2001), whereas a nearby, thirdepitope on this loop, defined by monoclonal antibody 2C1, was conserved.In none of the F-NDV mutant strains the point mutation Asp-72-Glyaffects the recognition of the antigenic site A5, I, 2, defined bymonoclontal antibody 12C4. Chen et al. (2001) showed based on theirthree-dimensional model of the F-protein that this epitope is determinedby surface-exposed residues at a distant loop segment, between strandsIf and Ig.

[0019] Finally, an Arg-101-Met substitution was observed in the HN andthe HN+F mutants, which did not result in an altered recognition by thetested monoclonal antibody in ELISA and neither did the mutations atamino acids 320 and 467 of the F-gene (Table 5), as they wereconservative.

[0020] Immunoselection with the HN-protein specific monoclonal antibody8C11 induced point mutations in the gene of this protein. Sequenceanalysis (Table 5) demonstrated Leu-193-Ser substitutions in the HN andHN+F strains, and a Leu-160-Gln substitution in the HN+F strain.Furthermore, a conservative ACA to ACG mutation is observed in codon 41in both these strains. None of these mutations result in the loss of anepitope recognized by HN-protein-specific MAb, as assessed by ELISA(Table 4).

[0021] However, the haemagglutination induced by the HN+F mutant is notinhibited by the monoclonal antibody 8C11, which was used for selection,indicating that the 8C11 epitope, although expressed, might not befunctionally active anymore. The haemagglutination mediated by the HNmutant is not inhibited, or reduced in comparison to the original LaSota strain, by several monoclonal antibodies. This possibly indicatesdefective expression and functioning of its entire HN molecule. Sequenceanalysis further demonstrated that the F and the derived F+HN mutant arecharacterized by Asn-115-Ser and Arg-124-Gly substitutions in theHN-protein. These mutations do not influence the recognition of the Fmutant by the examined monoclonal antibody in ELISA (Table 3) or thehaemagglutination inhibition assay (Table 4). The lack of reactivity ofthe monoclonal antibody 8C11 with the F+HN mutant in these assays musttherefore be entirely attributed to the observed Leu-229-Argsubstitution. Likewise, this substitution appears to induce therecognition of this La Sota mutant strain by a Hichner strain specificmonoclonal antibody 10B12 in the HI, but not in the ELISA assay.

[0022] According to an embodiment, the present invention relates to anattenuated mutant Newcastle disease La Sota virus strain as describedabove characterized in that its haemagglutination is not inhibited bymonoclonal antibody 8C11 which specifically recognises Newcastle diseasevirus glycoprotein HN.

[0023] Haemagglutination inhibition assays are well known in the art andallow to investigate the expression of functionally active epitopes onsaid virus strains. As mentioned before, the characterization of theattenuated mutant Newcastle disease La Sota virus strains F, HN, HN+Fand F+HN in such haemagglutination inhibition assays is described inexample 2 and Tables 2 and 3. With no inhibition of haemagglutination ismeant a signal less than 2.

[0024] According to another embodiment, the present invention alsorelates to an attenuated mutant Newcastle disease La Sota virus strainas defined above characterized in that it is not recognized bymonoclonal antibody 8C11 in an indirect ELISA assay, wherein saidmonoclonal antibody 8C11 specifically recognizes Newcastle disease virusglycoprotein HN. No binding to said antibody indicates that the finalsignal (O.D. or absorbance) obtained in the ELISA assay is less than0.120.

[0025] According to yet another embodiment, the present inventionfurther relates to an attenuated mutant Newcastle disease La Sota virusstrain as described above characterized in that it is not recognized bymonoclonal antibodies 1C3 or 10F2 in an indirect ELISA assay, whereinsaid monoclonal antibodies specifically recognize Newcastle diseasevirus glycoprotein F.

[0026] Indirect ELISA assays are well known in the art and binding ofthe virus strains HN, F, HN+F and F+HN in such an assay is given inexample 2 and Table 4.

[0027] According to yet another embodiment, the present invention alsorelates to an attenuated mutant Newcastle disease La Sota virus strainas described above and deposited as La Sota mutant 1C3+8C11, underregistration number CNCM I-2714, in the National Collection of Culturesof Microorganisms of the Pasteur institute in Paris. Surprisingly, saidattenuated mutant Newcastle disease La Sota virus strains have beenfound suitable for in ovo vaccination. This is further illustrated inexample 3. The pathogenicity of both the HN and the F virus strain wasreduced substantially in comparison with the parental lentogenic La Sotastrain from which they were derived. Moreover, the pathogenicity of boththe HN+F and the F+HN mutant strains was even more drastically reducedin comparison with the parental La Sota strain. Hatchability andneonatal survival were generally higher for chicks inoculated with thedouble mutant strains than with the F and HN strains.

[0028] In ovo vaccination involves the administration of said attenuatedvirus strains to eggs. Said eggs are fertile eggs which are preferablyin the fourth quarter of incubation. Chicken eggs are treated on aboutthe fifteenth to nineteenth day of incubation, and are most preferablytreated on about the eighteenth day of incubation. Turkey eggs arepreferably treated on about the twenty-first to twenty-sixth day ofincubation, and are most preferably treated on about the twenty-fifthday of incubation.

[0029] Eggs may be administered the vaccine of the invention by anymeans which transports the compound through the shell. The preferredmethod of administration is, however, by injection. The site ofinjection is preferably within either the region defined by the amnion,including the amniotic fluid and the embryo itself, in the yolk sac, orin the air cell. Most preferably, injection is made into the regiondefined by the amnion. By the beginning of the fourth quarter ofincubation, the amnion is sufficiently enlarged that penetration thereofis assured nearly all of the time when injection is made from the centerof the large end of the egg along the longitudinal axis.

[0030] The mechanism of injection is not particularly critical providedthat it does not unduly damage tissue and organs of the embryo. Forexample, a small hole is pierced with a needle (1-1{fraction (1/2)}inch, about 22 gauge) attached to syringe in the large end of the shelland the vaccine is injected below the inner shell membrane and thechorioallantoic membrane. Subsequently, the vaccinated embryonated eggsare transferred to an incubator to hatch. Several devices are availablefor said in ovo vaccination, exemplary being those disclosed in U.S.Pat. No. 4,681,063; U.S. Pat. No. 4,040,388; U.S. Pat. No. 4,469,047 andU.S. Pat. No. 4,593,646.

[0031] Although in ovo injection of the live virus strains according tothe present invention is preferable, these viruses can also beadministered by the mass application techniques commonly used for NDvaccination. These techniques include drinking water and sprayvaccination. Because of the extremely mild properties of the vaccine,spray administration of the vaccine is in particular contemplated.

[0032] The attenuated Newcastle disease La Sota virus strains of thepresent invention may be incorporated in a vaccine. Therefore, thepresent invention also relates to a vaccine composition which providesprotective immunity against Newcastle disease comprising an attenuatedmutant Newcastle disease La Sota virus strain as mentioned above.

[0033] In example 4 results are described illustrating thatadministration of mutant viruses to SPF embryos induces a virus specificimmune response.

[0034] The vaccine according to the invention may be prepared andmarketed in the form of a suspension or in a lyophillised form andadditionally contains a pharmaceutically acceptable carrier or diluentcustomary for such compositions. Carriers include stabilisers,preservatives and buffers. Suitable stabilisers are, for example SPGA,carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran,glutamate or glucose), proteins (such as dried milk serum, albumin orcasein) or degradation products thereof. Suitable buffers are forexample alkali metal phosphates. Suitable preservatives are thimerosal,merthuilate and gentamicin. Diluents include water, aqueous buffer (suchas buffered saline) and polyols (such as glycerol).

[0035] The composition of the present invention may also be used for inovo vaccination as a mixed vaccine in combination with at least onevaccine selected from the group consisting of vaccines to other virusessuch as e.g. avian infectious bronchitis virus, avian infectious bursaldisease virus, avian encephalomyelitis virus, egg drop syndrome virus,influenza virus, reovirus, adenovirus, etc.; bacteria such as e.g.Haemophilus paragallinarum, Salmonella typhimurium, S. enteritidis, S.pullorum, S. gallinarum, E. coli, Clostridium spp., Campylobacter spp.,Mycoplasma spp., etc.; and protozoans such as e.g. Eimeria tenella, E.maxima, E. acervulina, E. brunetti, E. necatrix, chicken malaria, etc.According to yet another aspect, the present invention relates to theuse of an attenuated mutant Newcastle disease La Sota virus strain ofthe invention as described above for the preparation of a vaccine for inovo vaccination of avian species against Newcastle disease.

[0036] According to another embodiment, the attenuated mutant Newcastledisease La Sota virus strain of the invention as described above mayalso be used for the preparation of a vaccine of avian species forpost-hatch application

[0037] According to yet another aspect, the present invention relates toa method for producing a vaccine for in ovo vaccination of avianspecies, said vaccine comprising attenuated mutant avian virus strains,which are selected using virus specific antibodies.

[0038] Thus, the invention relates to a method of producing virusstrains with reduced embryonic pathogenicity using a selection methodbased on specific antibodies or fragments thereof. By culturing virus inthe presence of antibodies, virus particles with an altered recognitionby the practiced antibodies may be selected. By repeating this selectionserially, antibody resistant virus strains may be obtained. Themultiplication of the selected virus between selections may be required.

[0039] The system to culture the virus in the presence of antibodies isnot critical. Virus culture systems may consist of entire, or parts oftissues, isolated avian or mammalian cells, or fertilized eggs. Cellsmay be primary cultures or established cell lines.

[0040] The antibodies used in practicing the present invention are virusspecific antibodies. Virus specific antibodies are those, which interactwith the virus if the virus and the antibodies are allowed to reacttogether for a sufficient time. The source of the virus specificantibodies is not critical. They may originate from any animal includingmammals (mouse, rat, rabbit) and birds (e.g. chicken, turkey).

[0041] The present method of the invention is particularly apparent inthe prevention of lethal diseases, which threaten birds early in life.One of the most prevalent and economically destructive diseases of thepoultry industry is Newcastle disease. However, the method of selectingattenuated mutant virus strains using virus specific antibodies may alsobe extended to other immunizable avian viral diseases.

[0042] The virus strains obtained by said immunoselection may bemultiplicated in a tissue culture system, such as an in vitro culture ofcells, or in an in vivo system, such as fertilized chicken eggs.

[0043] Subsequent screening by inoculation of avian embryos allowsselecting those virus strains with reduced pathogenicity. If theproduced virus strains induce an active immune response, which may beprotective, they may be used as vaccine. Based on differences in immuneresponses induced by the antigenic variants, produced as describedabove, subjects vaccinated with these variants may be discriminated. Anexample for this is the discrimination of chicks vaccinated with mutantNewcastle disease La Sota strains from chicks vaccinated with theparental La Sota strain based upon differences in the virus specificantibodies.

[0044] The principle of the preparation of attenuated Newcastle diseasevirus strains by immunoselection is explained in full detail inexample 1. In said example the selection is done using monoclonalantibodies 8C11 and IC3 directed against HN and F glycoproteins,respectively.

[0045] According to another embodiment, the present invention alsorelates to the method as described above wherein said virus specificantibodies are specific for viral avian diseases selected from the groupconsisting of Newcastle disease, infectious bronchitis, infectiousbursal disease, adenovirus diseases, reovirus, pox, laryngotracheitisand influenza.

[0046] Avian viruses causing said diseases are hereby included, such asavian herpesviruses (e.g. avian infectious laryngotracheitis, Marek'sdisease virus, . . . ), avian coronaviruses (e.g. avian infectiousbronchitis virus, turkey enteritis virus, . . . ), avian birnaviruses(e.g. infectious bursal disease virus), avian enteroviruses (e.g. avianencephalomyelitis virus), avian astroviruses, avian adenoviruses groupI, II and III, avian pneumoviruses (e.g. avian rhinotracheitis virus),avian reovirus (e.g. viral arthritis virus), avian circoviruses (e.g.chicken anemia virus) and avian poxviruses. In principle, the method canbe applied to all avian viruses for which neutralizing monoclonals orpolyclonal antiserum is available.

[0047] As mentioned above, in case of Newcastle disease, neutralizingantibodies which are directed against the For HN viral glycoprotein wereused in the examples described further below in this description.

[0048] Therefore, according to another embodiment, the present inventionalso relates to the method as mentioned above wherein said virusspecific antibodies specifically recognize an epitope on Newcastledisease virus glycoproteins HN and/or F.

[0049] Attenuated mutant Newcastle disease virus strains selected bysaid antibodies may be differentiated from the parental virus strainamong others by their reactivity with MAb in ELISA and inhaemagglutination assays or by the nucleotide sequence of their genes.The lack of neutralisation by homologous MAb after each passage inrelation to the non-treated control virus may be used as criterion todistinguish antigenic variant or mutant virus from revertant virus(Fleming et al. 1986).

[0050] According to another embodiment, the present invention alsorelates to the method as mentioned above wherein said virus specificantibodies specifically recognize an epitope on Newcastle disease LaSota virus glycoprotein HN.

[0051] According to yet another embodiment, the present inventionrelates to the method as mentioned above wherein said virus specificantibodies specifically recognize an epitope on Newcastle disease LaSota virus glycoprotein F.

[0052] According to yet another embodiment, said virus specificantibodies as mentioned above are monoclonal antibodies 8C11 or 1C3.

[0053] According to yet another aspect, the present invention alsorelates to a vaccine obtainable by the method according to the inventionsuitable for in ovo vaccination of avian species against viral diseases.

[0054] According to another aspect, the present invention relates to avaccine obtainable by the method according to the invention suitable forpost-hatch vaccination of avian species.

[0055] According to yet another aspect, the present invention relates tothe use of antibodies specifically recognizing an epitope of HN and/or Fglycoproteins or proteins similar thereto on lentogenous virus strainsfor selecting an attenuated mutant virus strain.

[0056] According to another embodiment, the invention relates to the useof antibodies as mentioned above specifically recognizing an epitope ofHN and/or F glycoproteins on Newcastle disease virus for selecting anattenuated mutant Newcastle disease virus strain.

[0057] According to yet another embodiment, the invention relates to theuse of antibodies as mentioned above specifically recognizing an epitopeon the HN or F glycoprotein of Newcastle disease La Sota virus forselecting an attenuated mutant La Sota virus strain.

[0058] According to yet another embodiment, the present inventionrelates to the use as mentioned above comprising the use of monoclonalantibodies 8C11 and/or 1C3. The following examples are intended only tofurther illustrate the invention and are not intended to limit the scopeof the invention, which is defined by the claims.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0059] Table 1. Neutralisation of antigenic variants afterneutralisation using homologous MAb.

[0060] Table 2. Characterisation of NDV La Sota strains byhaemagglutination inhibition assay using NDV specific MAb.

[0061] Table 3. Characterisation of NDV La Sota strains byhaemagglutination inhibition assay using NDV specific MAb.

[0062] Table 4. Reactivities of NDV-specific MAb with different NDVstrains in indirect ELISA.

[0063] Table 5. Sequence analysis of the genes coding for the F- andHN-glycoproteins of NDV La Sota strains

[0064] Table 6. Influence of inoculation at ED18 with different doses ofthe NDV La Sota strain on the hatchability of SPF eggs.

[0065] Table 7. Influence of inoculation at ED18 with different doses ofthe NDV La Sota HN strain (Exp. 1-3) and the F mutant strain (Exp. 4) onthe hatchability and neonatal survival.

[0066] Table 8. Influence of inoculation at ED18 with different doses ofthe NDV La Sota 1C3+8C11 (F+HN) strain on the hatchability and neonatalsurvival.

[0067] Table 9. Influence of inoculation at ED18 with different doses ofthe NDV La Sota double mutant strains on the hatchability of NDVnegative eggs.

[0068] Table 10. Influence of inoculation at ED18 with different dosesof the NDV La Sota HN+F strain on the hatchability of SPF eggs.

[0069] Table 11. Influence of inoculation at ED18 with different dosesof the NDV La Sota double mutant F+HN on the hatchability of eggs andpost hatch mortality of commercial broiler chickens.

[0070] Table 12. In ovo vaccination with indicated doses of the F+HNmutant strain and effect on survival of commercial broiler chickensafter intramuscular challenge with 10⁵ EID₅₀ of the Texas GB strain onday 43 post hatch.

[0071]FIG. 1 Mean NDV specific responses of SPF chickens (n=4) upon inovo vaccination with 100 EID50 of the La Sota HN mutant in function ofage FIG. 2 Mean NDV specific responses of SPF chickens upon in ovovaccination with 105 EID50 of the La Sota F+HN mutant in function ofage. For 4 and 17 day old chicks, n=4, for 14 and 21 day old chicks,n=3.

[0072]FIG. 3 Mean NDV specific responses of SPF chickens upon in ovovaccination with 102 EID50 of the La Sota F+HN mutant in function ofage. For 4 and 7 day old chicks, n=4, for 14 and 21 day old chicks, n=3.

[0073]FIG. 4 Mean NDV specific haemagglutination inhibition responsesupon in ovo vaccination with the La Sota F+HN mutant in function of age.Open symbols represent commercial broiler chickens, closed symbolsrepresent unvaccinated SPF chickens housed in the same isolator. Theresponses of the latter are a measure for dissemination of the vaccinevirus.

[0074]FIG. 5 Mean NDV specific IgG responses of commercial broilerchickens upon in ovo vaccination with the La Sota F+HN mutant infunction of age.

[0075]FIG. 6 Mean NDV specific IgM responses of commercial broilerchickens upon in ovo vaccination with the La Sota F+HN mutant infunction of age.

EXAMPLES Example 1 Preparation of Antibody Resistant NDV La Sota Strainsby Immunoselection

[0076] HN and F antigenic variant viruses were obtained from thelentogenic La Sota NDV strain by immunoselection, as described byMeulemans et al. (1987), using the MAb 8C11 and 1C3 directed againstthese two viral glycoproteins, respectively.

[0077] After culture on embryonic chicken hepatocytes in the presence ofone of these MAb, the antibody resistant virus was multiplied on 9 to 11day old chicken SPF embryos. This procedure was repeated four times,successively. After each passage, the neutralisation by homologous MAbwas examined and related to the non-treated control virus. Table 1 showsthat after 4 passages, the HN- and F-mutant strains show strongresistance to neutralisation. For these antigenic variants, the virustitre after neutralisation using the homologous antibodies nearlydiffered with less than 1 log 10 from the untreated controles. Thelatter criterion was defined by Fleming et al. (1986) as criterion todistinguish antigenic variant from revertant virus.

[0078] In addition so called double mutants were produced byimmunoselection as described above, using F and HN specific MAb 1C3 and8C11 starting from the HN and F mutant strains, respectively. Thesestrain were referred to as F+HN mutant, for the double mutant strainselected from the F mutant strain with the HN specific MAb 8C11, andHN+F mutant for the double mutant strain selected from the HN mutantstrain with the F specific MAb 1C3. Table 1 indicates that these mutantscan be regarded as true variant viruses on the basis of neutralisationresults, according to the criterion of Fleming (1986), because for eachof these antigenic variants the virus titre after neutralisation usingthe homologous MAb differed with less than 1 log 10 from the untreatedcontrol.

[0079] From Table 1, the frequency of an antibody resistant mutant inthe parental virus population can be estimated as the ratio of theTCID50/ml of the virus in the presence of MAb after the first passageand the corresponding TCID50/ml of the virus without MAb. Theapproximate frequencies of 1C3 resistant mutants in the parental La Sotastrain and the HN mutant strain are thus 10⁻5 and 10⁻⁶, respectively.The approximate frequencies of 8C11 resistant mutants in the parental LaSota strain and the HN mutant strain are 10⁻³ and 10⁻⁴, respectively.The frequency of finding the double mutants in the parental La Sotastrain would thus be 10⁻⁹ for both the F+HN and the HN+F mutant.

Example 2 Characterization of NDV La Sota Mutant Strains.

[0080] The produced La Sota strains were characterized based on theinhibition of haemagglutination by NDV specific MAb in two independentexperiments (Table 2 and Table 3). For all NDV strains tested,haemagglutination was not inhibited by MAb directed against the Fprotein of NDV, or by the MAb against cIFNγ which served as a negativecontrol. Likewise, haemagglutination was not inhibited by the MAbspecific for HN protein of the Ulster strain. Surprisingly, the MAb10B12 specific for the HN protein of the Hitchner strain inhibitedhaemagglutination induced by the F+HN strain. For the other NDV strainstested, haemagglutination was not inhibited by this MAb, as expected.

[0081] The HN specific MAb 8C11, 4D6, 6C6, 7B7 and 12B7 inhibitedhaemagglutination of the parent La Sota and the F mutant strain. The Fmutant strain can thus not be discriminated from the parental NDV LaSota strain in this haemagglutination inhibition assay. Further, for theF+HN and HN+F mutant strains, all these HN specific MAb except the MAb8C11 which was used for selection, inhibited haemagglutination. Thisindicates that in these double mutants, immunoselection with 8C11resulted in the loss of the 8C11 epitope, while all other epitopesexamined were conserved. Conversely, these HN specific MAb did virtuallynot (8C11, 4D6, 12B7 and 5A1) or only weakly (6C6, 7B7 and 7D4) inhibitthe haemagglutination induced by the HN mutant (Table 3), indicatingthat for this mutant immunoselection with 8C11 had an effect on a numberof epitopes spanning the entire HN molecule. Because thehaemagglutination inhibition test only allows determining the expressionof functionally active epitopes on the HN molecule, the expression ofNDV specific epitopes by the HN and F molecules of the different NDVstrains was assessed. For this, the binding of HN and F specific MAbwith different purified NDV strains coated to ELISA plates wasdetermined by indirect ELISA and quantified as absorbance. In general,the absorbances of the F+HN and the HN+F strains were lower than for theother strains, which most probably should be explained by a reducedamount of virus coated to the plate.

[0082] When this is taken in account, Table 4 shows that the reactivityof MAb remained unaltered where immunoselection was absent, i.e. in theHN and F proteins of respectively the F and HN mutant strains. Further,the F, F+HN and HN+F strains react identically with F specific MAb: inall these cases selection with the MAb 1C3 results thus in the loss ofthe epitopes seen by 1C3 and 10F2, while the epitopes seen by 2C1 and12C4 appear to be conserved.

[0083] Two different types of selection were however observed for theselection of mutants with the HN specific MAb 8C11. At one side, thereactivities of the HN mutant strain and the parental La Sota strainswith HN specific antibodies are identical, indicating that the HN, andalso the F protein, remained largely unaltered and that in this case,immunoselection with the HN specific MAb 8C11 did not result in thedisappearance of the epitope recognised by 8C11, or by any of the otherMAb. Also, the reactivity of the HN+F mutant strain with HN specificantibodies remained unchanged by this selection. This was expected asthe latter strain was derived from the HN strain. At the other side,selection of the F mutant with 8C11 to produce the F+HN mutant resultedin the loss of the 8C11 epitope, while the other HN specific epitopeswere conserved.

[0084] Combining Table 3 and Table 4 we can unambiguously characterizeall mutated NDV strains. The HN mutant conserved all epitopes examined,but the lack of inhibition of haemagglutination by several MAb indicatespossibly defective expression and functioning of the HN molecule. The Fmutant is characterized by a mutation in the F protein only. The HN+Fmutant also conserved all HN epitopes examined, but HI is onlyeliminated with the MAb 8C11 which was used for selection, indicatingthat the 8C11 epitope, although expressed is not functionally activeanymore. Its F protein has the same antibody reactivity as the otherstrains selected with the F specific MAb 1C3, namely the F and the F+HNmutant strain. Typical for the F+HN strain, besides the mutant Fprotein, is that it has structurally and functionally lost the 8C11epitope on the HN protein, whereas al other epitopes examined on thisprotein were conserved.

[0085] To further characterize all mutated NDV strains, the sequence ofthe genes encoding their HN and F proteins were sequenced as describedby Meulemans et al. (2001). Table 5 shows the detected mutations, incomparison with the parental La Sota strain. The sequence of the HN andF proteins of the latter was related to the sequences of these proteinspublished in the EMBL database under accession number AF077761 (La SotaNDV, complete genome).

Example 3 Reduction of the Pathogenicity of NDV La Sota Mutant Strainsfor Embryos

[0086] Table 6 shows that the La Sota NDV strain widely used for posthatch vaccination is toxic to embryos and unsuitable, in current form,for in ovo use. Even very low doses of virus strongly depressedhatchability, while the few hatched chicks were of poor quality showingsevere respiratory problems.

[0087] Table 7 demonstrates, although hatching percentages and neonatalsurvival tended to show high variability between groups, that thepathogenicity of both the HN and the F mutant for embryos and youngchicks was reduced substantially in comparison with the parental La Sotastrain. Indeed, Table 6 shows that inoculation of the latter strainresulted in 24 to 0% hatchability. Vaccination doses of 100 EID₅₀ orless tended to result in similar hatchability and neonatal survival inchicks treated with the HN mutant as in sham treated chicks.

[0088] In Table 8, Table 9 and Table 10 the influence of inoculation atED18 with different doses of the NDV LaSota F+HN mutant strain and theHN+F mutant strain on the hatchability and neonatal survival aresummarised. As for the F and HN mutant strains, hatching percentages andneonatal survival tended to show high variability between groups.However, it can be concluded that the pathogenicity of both the F+HN andthe HN+F mutants was drastically reduced in comparison with the parentalLa Sota strain (Table 6). Moreover, hatchability and neonatal survivalwere generally higher for chicks inoculated with the double mutantstrains than with the F and HN mutant strains.

Example 4 Administration of Mutant Viruses to SPF Embryos Induces aVirus Specific Immune Response

[0089] In ovo administration of 100 EID₅₀ of the La Sota HN mutant toSPF embryos results in the production of NDV specific IgM and IgGantibodies that inhibit haemagglutination (FIG. 1). These antibodytitres are of the same magnitude as the titres of age matched chicksreceiving 10⁵ EID₅₀ La Sota at hatch by eye drop vaccination (FIG. 1).

[0090] Likewise, in ovo administration of 10⁵ (FIG. 2) and 10² EID₅₀(FIG. 3) of the La Sota F+HN mutant in SPF embryos results in theproduction of NDV specific IgM and IgG antibodies that inhibithaemagglutination. Serum antibody titres appear independent of the dosestested because similar kinetics and IgM titres are observed for bothtreatments. IgG titres seem even higher for 10² EID₅₀ than for 10⁵EID₅₀, but this should be interpreted cautiously because of the lownumber of chicks examined. (FIG. 2+3)

Example 5 Embryonic Administration of Mutant Viruses Induces a VirusSpecific Protective Immune Response in the Presence of MaternalAntibodies

[0091] In agreement with example 3, in ovo administration of the F+HNmutant to 18 day old embryos of commercial broilers did not result in asignificant reduction of the hatchability (Table 11). Further, chicksreceiving 0, 10² and 10³ EID50 showed no or low post hatch mortality. Inthe isolator containing most birds, 6 of 46 chicks receiving 104 EID50died for unknown reasons. These mortalities occurred later than expectedfor possible virus induced mortality (days 8, 10, 11, 14, 18, 21 and 36)while no clinical symptoms, except feather picking were observed.

[0092] An NDV specific IgM response, which peaked around 14 days of age,is observed in chicks vaccinated at embryonic day 18 with 10³ and 10⁴EID₅₀ of the F+HN mutant, but not in PBS treated chicks and in chicksreceiving 10² EID₅₀ of this mutant (FIG. 6). In the former chicks, NDVspecific IgG, and HI titers, gradually increases with age, indicatingisotype switching and an active production of NDV specific IgG (FIG. 5).In the latter groups, HI titers and NDV specific IgG of maternal origingradually decrease to reach background levels around 3 weeks of age.Maternal NDV specific antibodies are thus not replaced by NDV specificantibodies produced by the chicks themselves.

[0093] HI titres (FIG. 4) and NDV specific IgM and IgG (not shown) werefound in the serum of 14 and 21 day old SPF chicks (sentinels) housed inthe same isolators as the chicks inoculated with 10³ and 10⁴ EID₅₀ ofthe F+HN strain, but not in the serum of 14 and 21 day old SPF chicks(sentinels) housed in the same isolators as the control chicks and thechicks inoculated with 10² EID₅₀ of the F+HN strain. This demonstratesthat, if adequate doses are administered, the vaccine virus proliferatesin the presence of maternal antibodies and is disseminated fromvaccinated to non-vaccinated chicks

[0094] The induction of NDV specific antibodies correlates well withprotection against challenge with the very virulent TexasGB strain.Indeed, all chicks were protected against intramuscular challenge with10⁵ EID₅₀ of the Texas GB strain in those groups (receiving 10³ or 10⁴EID₅₀) where virus specific antibodies were detected in the serum (Table12). On the contrary, if no antibodies were present (control chicks andchicks receiving 10² EID₅₀ of F+HN), all chicks died or were moribund.An increase of NDV specific IgM, IgG and HI titres was observed in thechicks surviving challenge (FIG. 4, FIG. 5 and FIG. 6). TABLE 1Neutralisation of antigenic variants after neutralisation usinghomologous MAb Virus titre after passage (TCID₅₀/ml) Treatment 1^(st)passage* 2^(nd) passage 3^(rd) passage 4^(th) passage F mutant MAb 1C3  2.13 * 10⁴ >1.58 * 10⁹ 1.20 * 10⁹ 1.58 * 10⁸ No MAb >1.58 *10⁹ >1.58 * 10⁹ 2.39 * 10⁹ 2.13 * 10⁹ HN mutant MAb 8C11   1.58 * 10⁶  1.20 * 10⁶ 1.58 * 10⁷ 1.58 * 10⁶ No MAb >1.58 * 10⁹ >1.58 * 10⁹ 2.39 *10⁸   5 * 10⁷ F + HN mutant MAb 8C11   2.13 * 10⁵   3.38 * 10⁵ 9.96 *10⁸ No MAb   1.58 * 10⁹   3.38 * 10⁸   5 * 10⁸ HN + F mutant MAb 1C3  1.58 * 10²   7.39 * 10⁷ 2.13 * 10⁷ No MAb   1.58 * 10⁸   2.39 * 10⁸7.39 * 10⁷

[0095] TABLE 2 Characterisation of NDV La Sota strains byhaemagglutination inhibition assay using NDV specific MAb SpecificityMAb La Sota F HN F + HN HN + F HN protein 8C11  12* 12 1 3 3 4D6 12 12 612 12 6C6 12 12 9 12 12 7B7 12 12 10 12 12 12B7 12 12 5 12 10 IFN γ  1 12 1 1

[0096] TABLE 3 Characterisation of NDV La Sota strains byhaemagglutination inhibition assay using NDV specific MAb SpecificityMAb La Sota F HN F + HN HN + F HN protein 8C11     6* 7 <2 <2 <2 4D6  11 >12 <2 >12 10 6C6   10 11 7 >12 11 7B7   10 >12 8 >12 11 12B7   1011 <2 11 10 La Sota (HN) 7D4   10 >12 7 >12 10 Lentogenous (HN) 5A1   1111 3 >12 11 Ulster Italien (HN) 3C5  <2 <2 <2 <2 <2 Hitchner (HN) 10B12 <2 2 <2 5 2 F protein 1C3  <2 <2 <2 <2 <2 2C1  <2 <2 <2 <2 <2 10F2  <2<2 <2 <2 <2 12C4  <2 <2 <2 <2 <2 Anti cIFN γ  <2 <2 <2 <2 <2 Positivecontrol PAb    8 10 8 10 11

[0097] TABLE 4 Reactivities of HN specific MAb with different NDVstrains in indirect ELISA Specificity MAb La Sota F HN F + HN HN + F HNprotein 8C11 1.049* 1.164 1.015 0.049 0.514 4D6 1.59 1.593 1.475 0.6880.705 6C6 1.323 1.291 1.113 0.492 0.616 7B7 1.429 1.416 1.272 0.5050.557 12B7 1.55 1.533 1.314 0.535 0.672 La Sota (HN) 7D4 1.52 1.4751.415 0.536 0.676 Lentogenous (HN) 5A1 1.264 1.234 1.11 0.533 0.579Hitchner (HN) 10B12 1.084 1.075 0.97 0.401 0.511 F protein 1C3 0.8080.03 0.779 0.047 0.104 2C1 0.932 0.597 0.905 0.76 0.7 10F2 0.481 0.0280.504 0.039 0.046 12C4 0.805 0.528 0.714 0.848 0.593 Anti cIFN γ 0.0290.035 0.038 0.039 0.058 Positive control PAb 2 1.931 1.993 1.803 1.737

[0098] TABLE 5 Sequence analysis of the genes coding for the F- and HN-glycoproteins of NDV La Sota strains La Sota AF077761^(b) F F + HN HNHN + F F-gene   72^(a) GAT (Asp) TAT (Tyr) TAT (Tyr) GAA (Glu) 101 AGG(Arg) ATG (Met) ATG (Met) 320 CCA (Pro) CCC (Pro) 467 CTC (Leu) CTT(Leu) CTT(Leu) CTT (Leu) CTT (Leu) CTT (Leu) HN-gene  41 ACA (Thr) ACG(Thr) ACG (Thr) 115 AAT (Asn) AGT (Ser) AGT (Ser) 124 AGG (Arg) GGG(Gly) GGG (Gly) 158 GAG (Glu) GAA (Glu) 160 CTG (Leu) CAG (Gln) 193 TTG(Leu) TCG (Ser) TCG (Ser) 229 CTG (Leu) CGG (Arg) 416 CGG (Arg) CGA(Arg) 508 AGC (Ser) GGC (Gly)

[0099] TABLE 6 Influence of inoculation at ED18 with different doses ofthe NDV La Sota strain on the hatchability of SPF eggs NDV strain Dose(EID₅₀) Total Hatched La Sota 1000 17  0 (0%) 100 17  2 (12%) 10 17  0(0%) 1 17  4 (24%) Control 0 16 13 (81%)

[0100] TABLE 7 Influence of inoculation at ED18 with different doses ofthe NDV La Sota HN mutant strain (Exp. 1-3) and the F mutant strain(Exp. 4) on the hatchability and neonatal survival Global Dose Neonatalsurvival Experiment (EID₅₀) Eggs Hatched survival (10 d) (10 d) 1 (HNmutant) 100000 18  3 (17%) N.D. 10000 18  2 (11%) N.D. 1000 17  3 (18%)N.D. 100 17  8 (47%) N.D. 0 21 11 (52%) N.D. 2 (HN mutant) 1000 21 16(76%) 10/16 (62%) 48% 100 21  5 (24%)  3/5 (60%) 14% 10 21 15 (71%)13/15 (87%) 62% 1 20 11 (55%) 10/11 (91%) 50% 0 21 13 (62%) 10/13 (77%)47% 3 (HN mutant) 200 18 11 (61%) 10/11 (91%) 55% 100 18 16 (89%) 16/16(100%) 89% 50 18 13 (72%) 13/13 (100%) 72% 25 18 16 (89%) 16/16 (100%)89% 12.5 18 14 (78%) 10/13 (77%) 55% 0 18 15 (83%) 15/15 (100%) 83% 4 (Fmutant) 1000 15 10 (67%)  3/10 (30%) 20% 100 15  7 (47%)  3/7 (43%) 20%10 15 11 (73%)  4/11 (36%) 27% 1 15  8 (53%)  4/8 (50%) 27% 0 15 12(80%) 10/12 (83%) 67%

[0101] TABLE 8 Influence of inoculation at ED18 with different doses ofthe NDV La Sota 1C3 + 8C11 mutant (F + HN) strain on the hatchabilityand neonatal survival Neonatal Ex- Dose survival Global survivalperiment (EID₅₀) Eggs Hatched (10 d) (10 d) Experi- 10⁵ 20  18 (90%)N.D.^(a) N.D. ment 1 10⁴ 20  14 (70%) 13/14 (93%) 13/20 (65%) 10³ 20  14(70%) 13/14 (93%) 13/20 (65%) 10² 20  17 (85%) 13/17 (76%) 13/20 (65%)10 20  17 (85%) 13/17 (76%) 13/20 (65%)  0  20  11 (55%)  9/11 (81%) 9/20 (45%) Experi- 10⁶ 12   7 (58%)  4/7 (57%)  4/12 (33%) ment 2 10⁵12  11 (92%)  7/11 (64%)  7/12 (58%) 10⁴ 12   9 (75%)  7/9 (78%)  7/12(58%) 10³ 12  12 (100%) 10/12 (83%) 10/12 (83%)  0  14  12 (86%) 11/12(92%) 11/12 (92%) Experi- 10⁴ 36 18^(b) (50%) 16/18 (90%) 16/36 (44%)ment 3 10³ 36  27 (75%) 24/27 (89%) 24/36 (66%)  0  36  31 (86%) 30/31(97%) 30/36 (83%) Experi- 10⁴ 28 16^(c) (73%) 10/16 10/28 (36%) ment 410³ 28  22 (78%) 18/22 18/28 (64% 10² 28 19^(d) (68%) 18/19 18/28 (64%) 0  28  22 (78%) N.D. N.D.

[0102] TABLE 9 Influence of inoculation at ED18 with different doses ofthe NDV La Sota double mutant strains on the hatchability of NDVnegative eggs Mutant Neonatal virus Dose survival strain (EID₅₀) TotalHatched (10 d) (F+HN) 10⁶ 9 3 (33%)^(a) 1/3 (33%) 10⁵ 9 5 (56%) 3/5(60%) 10⁴ 9 7 (78%) 5/7 (71%) 10³ 9 6 (67%) 3/6 (50%) (HN+F) 10⁶ 9 2(22%) 1/2 (50%) 10⁵ 9 2 (22%) 1/2 (50%) 10⁴ 9 7 (78%) 4/7 (57%) 10³ 9 3(33%) 3/9 (33%) Control  0  9 7 (78%) 7/7 (100%)

[0103] TABLE 10 Influence of inoculation at ED18 with different doses ofthe NDV La Sota HN + F strain on the hatchability of SPF eggs DoseNeonatal (EID₅₀) Total Hatched survival (10 d) 10⁶ 12   5 (42%)  1/5(20%) 10⁵ 12   5 (42%)  3/5 (60%) 10⁴ 12   4 (25%)  2/4 (50%) 10³ 12  10(83%)  8/10 (80%)  0  14  12 (86%) 11/12 (92%) 10⁵ 19  11 (58%) 10⁴ 19 12 (63%) 10³ 19  17 (89%) 10² 19 12^(a) (63%)  0¹¹ 20  17 (85%)

[0104] TABLE 11 Influence of inoculation at ED18 with different doses ofthe NDV La Sota double mutant F+HN on the hatchability of eggs and posthatch mortality of commercial broiler chickens Mutant Unexplained virusDose post hatch strain (EID₅₀ ) Total Hatched mortality (F + HN) 10⁴ 5146 (90%) 6/46 10³ 51 41 (80%) 1 10² 51 41 (80%) 0 Control  0  51 42(82%) 1

[0105] TABLE 12 In ovo vaccination with indicated doses of the F + HNmutant strain and effect on survival of commercial broiler chickensafter intramuscular challenge with 10⁵ EID₅₀ of the Texas GB strain onday 43 post hatch. Mortality by day 53 Treatment n % PBS treated 11/12^(a) 92 10² EID₅₀ F + HN 12/12 100 10³ EID₅₀ F + HN  0/12 0 10⁴EID₅₀ F + HN 0/9 0

REFERENCES

[0106] Abenes G, Kida H, Yanagawa R. (1986) Antigenic mapping andfunctional analysis of the F protein of Newcastle disease virus usingmonoclonal antibodies. Arch Virol 90: 97-110

[0107] Ahmad J, Sharma J M (1992) Evaluation of a modified-live virusvaccine administered in ovo to protect chickens against Newcastledisease. Am J Vet Res: 53: 1999-2004.

[0108] Ahmad J, Sharma J M (1993) Protection against hemorrhagicenteritis and Newcastle disease in turkeys by embryo vaccination withmonovalent and bivalent vaccines. Avian Dis 37:485-491

[0109] Chen L, Gorman J J, McKimm-Breschkin J, Lawrence L J, Tulloch PA, Smith B J, Colman P M, Lawrence M C (2001) The structure of thefusion glycoprotein of Newcastle disease virus suggests a novel paradigmfor the molecular mechanism of membrane fusion. Structure 9: 255-266

[0110] Birrer M J, Udem S, Nathenson S, Bloom B R. (1981) Antigenicvariants of measles virus. Nature 293: 67-69

[0111] EP 0848956 A1 (1998) In ovo vaccination against Newcastle disease

[0112] EP 1 074 614 A1 Mebatsion, (2001) A recombinant Newcastle diseasevirus for in ovo vaccination

[0113] EP0583998 Benejean J, Tuffereau M C, Coulon P, Flamand A, Lafay F(1994) Avirulent antirabies vaccine

[0114] Fleming J O, Trousdale M D, el-Zaatari F A, Stohlman S A, WeinerL P (1986) Pathogenicity of antigenic variants of murine coronavirus JHMselected with monoclonal antibodies. J Virol 58: 869-75

[0115] Gerhard W, Webster R G. (1978) Antigenic drift in influenza Aviruses. I. Selection and characterization of antigenic variants ofA/PR/8/34 (HON1) influenza virus with monoclonal antibodies. J Exp Med148: 383-92.

[0116] Haddad E E, Whiffill C E, Avakian A P, Ricks C A, Andrews P D,Thoma J A, Wakenell P S (1997). Efficacy of a novel infectious bursaldisease virus immune complex vaccine in broiler chickens. Avian Dis 41:882-889.

[0117] Long Le, Brasseur R, Wemers C, Meulemans G, Burny A (1988).Fusion (F) protein gene of Newcastle disease virus: sequence andhydrophobicity comparative analysis between virulent and avirulentstrains. Virus Genes 1: 333-50.

[0118] Long Le, Portetelle D, Ghysdael J, Gonze M, Burny A, Meulemans G(1986) Monoclonal antibodies to hemagglutinin-neuramimidase and fusionglycoproteins of Newcastle disease virus: relationship betweenglycosylation and reactivity. J Virol 57: 1198-202.

[0119] Lubeck M D, Schulman J L, Palese P. (1980) Antigenic variants ofinfluenza viruses: marked differences in the frequencies of variantsselected with different monoclonal antibodies. Virology 102: 458-462

[0120] Meulemans G, Boschmans M, Decaestesstecker M, van den Bergh T P,Denis P, Cavanagh D (2001) Epidemiology of infectious bronchitis virusin Belgian broilers: a retrospective study, 1986 to 1995. Avian Path 30:411-421

[0121] Meulemans G, Gonze M, Carlier M C, Petit P, Burny A, Le Long(1987b) Evaluation of the use of monoclonal antibodies to hemagglutininand fusion glycoproteins of Newcastle disease virus for virusidentification and strain differentiation purposes. Arch Virol 92: 55-62

[0122] Meulemans G, Gonze M, Carlier M C, Petit P, Burny A, Long Le(1987) Pathogenicity of antigenic variants of Newcastle disease virusItalian strain selected with monoclonal antibodies. Ann Rech Vet 18:371-474

[0123] Meulemans G, Gonze M, Petit P, Long Le, Burny A (1986) Protectiveeffects of HN & F glycoprotein-specific monoclonal antibodies onexperimental Newcastle disease. Avian Path. 15: 761-768

[0124] Meulemans G, Roels S, van den Bergh T P, Godfroid G, DeCaesteckere M (1998) Acute pancreatitis in chickens due to nonvirulentNewcastle disease virus. Vet Rec 143: 300-302

[0125] Neyt C, Geliebter J, Slaoui M, Morales D, Meulemans G, Burny A(1989) Mutations located on both F1 and F2 subunits of the Newcastledisease virus fusion protein confer resistance to neutralization withmonoclonal antibodies. J Virol 63: 952-954

[0126] Reddy S K, Sharma J M, Ahmad J, Reddy D N, McMillen J K, Cook SM, Wild M A, Schwartz R D (1996). Protective efficacy of a recombinantherpesvirus of turkeys as an in ovo vaccine against Newcastle andMarek's diseases in specific-pathogen-free chickens. Vaccine 14: 469-477

[0127] Ricks C A, Avakian A, Bryan T, Gildersleeve R, Haddad E, Ilich R,King S, Murray L, Phelps P, Poston R, Whitfill C, Williams C. (1999) Inovo vaccination technology. Adv Vet Med 41: 495-515

[0128] Russell P H, Alexander D J (1983) Antigenic variation ofNewcastle disease virus strains detected by monoclonal antibodies. ArchVirol 75: 243453

[0129] Sharma G, Greer W, Gildersleeve R P, Murray D L, Miles A M(1995). Field safety and efficacy of in ovo administration of HVT+SB-1bivalent Marek's disease vaccine in commercial broilers. Avian Dis 39:211-217

[0130] Sharma J M (1985). Embryo vaccination with infectious bursaldisease virus alone or in combination with Marek's disease vaccine.Avian Diseases 29: 1155-1169

[0131] Sharma J M (1986). Embryo vaccination of specific-pathogen-freechickens with infectious bursal disease virus: tissue distribution ofthe vaccine virus and protection of hatched chickens against disease.Avian Dis 30: 776-780

[0132] Sharma J M (1987). Delayed replication of Marek's disease virusfollowing in ovo inoculation during late stages of embryonaldevelopment. Avian Diseases 31(3):570-6.

[0133] Sharma J M (1999). Introduction to poultry vaccines and immunity.Adv Vet Med 41:481-94.

[0134] Sharma J M, Burmester BR. (1982). Resistance to Marek's diseaseat hatching in chickens vaccinated as embryos with the turkeyherpesvirus. Avian Dis 26:134-149.

[0135] Sharma J M, Witter R L (1983). Embryo vaccination against Marek'sdisease with serotypes 1, 2 and 3 vaccines administered singly or incombination. Avian Dis 27: 453-463

[0136] Stone H, Mitchell B, Brugh M (1997). In ovo vaccination ofchicken embryos with experimental Newcastle disease and avian influenzaoil-emulsion vaccines. Avian Diseases 41: 856-863

[0137] Toyoda T, Gotoh B, Sakaguchi T, Kida H, Nagai Y (1988)Identification of amino acids relevant to three antigenic determinantson the fusion protein of Newcastle disease virus that are involved infusion inhibition and neutralization. J Virol 62: 4427-4430

[0138] U.S. Pat. No. 4,040,388 Miller G E (1977) Method and apparatusfor automatic egg injection

[0139] U.S. Pat. No. 4,469,047 Miller G E (1984) Apparatus and methodfor injecting eggs

[0140] U.S. Pat. No. 4,593,646 Miller G E, Sheeks P (1986) Egg injectionmethod and apparatus

[0141] U.S. Pat. No. 4,681,063 Hebrank J H (1987) High speed automatedinjection system for avian embryos

[0142] U.S. Pat. No. 5,871,748 Thaxton J P, Tyczkowski J K, Thoma J A,Fredericksen T L, Whithfill C E (1999) Method of treating viral diseasesin animals

[0143] U.S. Pat. No. 5,427,791 (1995) Ahmad J, Sharma J, Embryonalvaccination of fowl against Newcastle disease is accomplished with ethylmethane sulfonate modified NDV-B1 virus

[0144] U.S. Pat. No. 6,032,612 Williams DJ (2000) Automated in ovoinjection apparatus

[0145] Wakenell P S, Sharma J M, (1986). Chicken embryonal vaccinationwith avian infectious bronchitis virus. Am J Vet Res 47: 933-938

[0146] Wakenell P S, Sharma J M, Slocombe R F (1995). Embryo vaccinationof chickens with infectious bronchitis virus: histologic andultrastructural lesion response and immunologic response to vaccination.Avian Dis 39: 752-765

[0147] Whitfill C E, Haddad E E, Ricks C A, Skeeles J K, Newberry L A,Beasley J N, Andrews P D, Thoma J A, Wakenell P S, (1995) Determinationof optimum formulation of a novel infectious bursal disease virus (IBDV)vaccine constructed by mixing bursal disease antibody with IBDV. AvianDis 39: 687-699

[0148] Whitfill C E, Ricks C A, Haddad E E, Andrews P A, Skeeles J K(1992) Infectious bursal disease (IBD) vaccine for day of ageadministration in broiler chickens. Poult Sci 71, 59

[0149] Yusoff K, Nesbit M, McCartney H, Meulemans G, Alexander D J,Collins M S, Emmerson P T, Samson A C (1989) Location of neutralizingepitopes on the fusion protein of Newcastle disease virus strainBeaudette C. J Gen Virol 70: 3105-3109.

1. An attenuated mutant Newcastle disease La Sota virus strain suitablefor in ovo vaccination of avian species comprising a mutation in thegene sequences encoding the HN and/or F glycoproteins of said virusresulting in an altered expression of said glycoproteins.
 2. Anattenuated mutant Newcastle disease La Sota virus strain according toclaim 1 characterized in that haemagglutination is not inhibited bymonoclonal antibody 8C11 which specifically recognizes Newcastle diseasevirus glycoprotein HN.
 3. An attenuated mutant Newcastle disease La Sotavirus strain according to claim 1 characterized in that it is notrecognized by monoclonal antibody 8C11 in an indirect ELISA assay,wherein said monoclonal antibody 8C11 specifically recognizes Newcastledisease virus glycoprotein HN.
 4. An attenuated mutant Newcastle diseaseLa Sota virus strain according to claim 1 characterized in that it isnot recognized by monoclonal antibodies 1C3 or 10F2 in an indirect ELISAassay, wherein said monoclonal antibodies specifically recognizeNewcastle disease virus glycoprotein F.
 5. An attenuated mutantNewcastle disease La Sota virus strain according to claim 1 chosen fromthe strains as deposited as La Sota mutant 1C3+8C11, under registrationnumber CNCM I-2714, in the National Collection of Cultures ofMicroorganisms of the Pasteur institute in Paris.
 6. A vaccinecomposition which provides protective immunity against Newcastle diseasecomprising an attenuated mutant Newcastle disease La Sota virus strainaccording to claim
 1. 7. Use of an attenuated mutant Newcastle diseaseLa Sota virus strain according to claim 1 for the preparation of avaccine for in ovo vaccination of avian species against Newcastledisease.
 8. Use of an attenuated mutant Newcastle disease La Sota virusstrain according to claim 1 for the preparation of a vaccine forpost-hatch vaccination against Newcastle disease.
 9. A method forproducing a vaccine for in ovo vaccination of avian species, saidvaccine comprising attenuated mutant avian virus strains which areselected using virus specific antibodies.
 10. The method according toclaim 9 wherein said virus specific antibodies are specific for viralavian diseases selected from the group consisting of Newcastle disease,infectious bronchitis, infectious bursal disease, adenovirus diseases,reovirus, pox, laryngotracheitis and influenza.
 11. The method accordingto claim 9 wherein said virus specific antibodies specifically recognizean epitope on Newcastle disease virus glycoproteins HN and/or F.
 12. Themethod according to claim 9 wherein said virus specific antibodiesspecifically recognize an epitope on Newcastle disease La Sota virusglycoprotein HN.
 13. The method according to claim 9 wherein said virusspecific antibodies specifically recognize an epitope on Newcastledisease La Sota virus glycoprotein F.
 14. The method according to claim9 wherein said virus specific antibodies are monoclonal antibodies 8C11or 1C3.
 15. A vaccine obtainable by the method according to claim 9suitable for in ovo vaccination of avian species against viral diseases.16. A vaccine obtainable by the method according to claim 9 suitable forpost-hatch vaccination of avian species against viral diseases.
 17. Useof antibodies specifically recognizing an epitope of HN and/or Fglycoproteins or proteins similar thereto on lentogenous virus strainsfor selecting an attenuated mutant virus strain.
 18. Use according toclaim 17 of antibodies specifically recognizing an epitope of HN and/orF glycoproteins on Newcastle disease virus for selecting an attenuatedmutant Newcastle disease virus strain.
 19. Use according to claim 18 ofantibodies specifically recognizing an epitope on the HN or Fglycoprotein of Newcastle disease La Sota virus for selecting anattenuated mutant La Sota virus strain.
 20. Use according to claim 19comprising the-use of monoclonal antibodies 8C11 and/or 1C3.